Neurotoxin implant

ABSTRACT

A biocompatible implant for continuous in vivo release of a neurotoxin over a treatment period extending from one month to five years. The implant can be made of casting a solution of a polymer, such as an ethyl vinyl acetate copolymer and the neurotoxin. The neurotoxin can be a botulinum toxin.

BACKGROUND

[0001] The present invention relates to a controlled release drugdelivery system. In particular, the present invention relates to acontrolled release neurotoxin delivery system.

[0002] A controlled release system can deliver a drug in vivo at apredetermined rate over a specific time period. Generally, release ratesare determined by the design of the system, and can be largelyindependent of environmental conditions such as pH. Controlled releasesystems which can deliver a drug over a period of several years areknown. Contrarily, sustained release systems typically deliver drug in24 hours or less and environmental factors can influence the releaserate. Thus, the release rate of a drug from an implanted controlledrelease system (an “implant”) is a function of the physiochemicalproperties of the carrier implant material and of the drug itself.Typically, the implant is made of an inert material which elicits littleor no host response.

[0003] A controlled release system can be comprised of a drug with abiological activity incorporated into a carrier. The carrier can be apolymer or a bioceramic material. The controlled release system can beinjected, inserted or implanted into a selected location of a patient'sbody and reside therein for a prolonged period during which the drug isreleased by the implant in a manner and at a concentration whichprovides a desired therapeutic efficacy.

[0004] Polymeric materials can release drugs due to diffusion, chemicalreaction or solvent activation, as well as upon influence by magnetic,ultrasound or temperature change factors. Diffusion can be from areservoir or matrix. Chemical control can be due to polymer degradationor cleavage of the drug from the polymer. Solvent activation can involveswelling of the polymer or an osmotic effect. See e.g. Science 249;1527-1533:1990.

[0005] A membrane or reservoir implant depends upon the diffusion of abioactive agent across a polymer membrane. A matrix implant is comprisedof a polymeric matrix in which the bioactive agent is uniformlydistributed. Swelling-controlled release systems are usually based onhydrophilic, glassy polymers which undergo swelling in the presence ofbiological fluids or in the presence of certain environmental stimuli.

[0006] Preferably, the implant material used is substantially non-toxic,non-carcinogenic, and non-immunogenic. Suitable implant materialsinclude polymers, such as poly(2-hydroxy ethyl methacrylate) (p-HEMA),poly(N-vinyl pyrrolidone) (p-NVP)+, poly(vinyl alcohol) (PVA),poly(acrylic acid) (PM), polydimethyl siloxanes (PDMS), ethylene-vinylacetate (EVAc) copolymers, polyvinylpyrrolidone/methylacrylatecopolymers, polymethylmethacrylate (PMMA), poly(lactic acid) (PLA),poly(glycolic acid) (PGA), polyanhydrides, poly(ortho esters), collagenand cellulosic derivatives and bioceramics, such as hydroxyapatite(HPA), tricalcium phosphate (TCP), and aliminocalcium phosphate (ALCAP).Lactic acid, glycolic acid and collagen can be used to makebiodegradable implants.

[0007] Controlled release systems comprising a polymer for prolongeddelivery of a therapeutic drug are known. For example, a subdermalreservoir implant comprised of a nonbiodegradable polymer can be used torelease a contraceptive steroid, such as progestin, in amounts of 25-30mg/day for up to sixty months (i.e. the Norplant® implant).Additionally, Dextran (molecular weight about 2 million) has beenreleased from implant polymers.

[0008] An implant material can be biodegradable or bioerodible. Anadvantage of a bioerodible implant is that is does not need to beremoved from the patient. A bioerodible implant can be based upon eithera membrane or matrix release of the bioactive substance. Biodegradablemicrospheres prepared from PLA-PGA are known for subcutaneous orintramuscular administration.

[0009] A degradable implant preferably retains its structural integritythroughout its duration of controlled release so that it can be removedif removal is desired or warranted. After the incorporated drug fallsbelow a therapeutic level, a biodegradable implant can degradecompletely without retaining any drug which can be released at lowlevels over a further period. Subdermal implants and injectablemicrospheres made of degradable materials, such as lactic acid-glycolicacid copolymers, polycaprolactones and cholesterol, for steroiddelivery, are known.

[0010] Protein Implants

[0011] Controlled release systems for large macromolecules, such asproteins are known. Thus, biocompatible, polymeric pellets whichincorporate a high molecular weight protein have been implanted andshown to exhibit continuous release of the protein for periods exceeding100 days. Various labile, high molecular weight enzymes (such asalkaline phosphatase, molecular weight 88 kD and catalase, molecularweight 250 kD) have been incorporated into biocompatible, polymericimplants with long term, continuous release characteristics. Generallyan increase in the polymer concentration in the casting solutiondecreases the initial rate at which protein is released from theimplant. Nature 263; 797-800:1976.

[0012] Additionally, albumin can be released from an EVAc implant andpolylysine can be released from collagen based microspheres.Mallapragada S. K. et al, at page 431 of chapter 27 in Von Recum, A. F.Handbook of Biomaterials Evaluation, second edition, Taylor & Francis(1999). Additionally, the release of tetanus toxoid from microsphereshas been studied. lbid at 432. Sintered EVAc copolymer insertedsubcutaneously has been shown to release insulin over a period of 100days. lbid at 433.

[0013] Furthermore, it is known to encapsulate a protein, such as humangrowth hormone (hGH) (molecular weight about 26 kD), within a polymericmatrix which when implanted permits the human growth hormone to bereleased in vivo over a period of about a week. U.S. Pat. No. 5,667,808.

[0014] A controlled release system (i.e. an “implant”) can exhibit ahigh initial burst of protein release, followed by minimal releasethereafter. Unfortunately, due to the high concentration of proteinwithin a controlled release matrix, the protein molecules tend toaggregate and form denatured, immunogenic concentrations of protein.

[0015] Pulsatile Release Implants

[0016] Hydrogels have been used to construct single pulse and multiplepulse drug delivery implants. A single pulse implant can be osmoticallycontrolled or melting controlled. Doelker E., Cellulose Derivatives, AdvPolym Sci 107; 199-265:1993. It is known that multiple pulses of certainsubstances from an implant can be achieved in response to anenvironmental change in a parameter such as temperature (Mater Res SocSymp Proc, 331;211-216:1994; J. Contr Rel 15;141-152:1991), pH (MaterRes Soc Symp Proc, 331;199-204:1994), ionic strength (React Polym,25;127-137:1995), magnetic fields (J. Biomed Mater Res,21;1367-1373:1987) or ultrasound.

[0017] Unfortunately, a subcutaneous implantable drug pellet made of anonbiodegradable polymer has the drawback of requiring both surgicalimplantation and removal. Use of a biocompatible, bioerodible implantcan overcome the evident deficiencies of nonbiodegradable implants. Abiodegradable implant can release a drug over a long period of time withsimultaneous or subsequent degradation of the polymer within the tissueinto constituents, thereby avoiding any need to remove the implant. Seee.g. Drug Development and Industrial Pharmacy 24(12);1129-1138:1998.

[0018] A degradable polymer can be a surface eroding polymer, as opposedto a polymer which displays bulk or homogenous. A surface erodingpolymer degrades only from its exterior surface, and drug release istherefore proportional to the polymer erosion rate. A suitable suchpolymer can be a polyanhydride.

[0019] Botulinum Toxin

[0020] The anaerobic, gram positive bacterium Clostridium botulinumproduces a potent polypeptide neurotoxin, botulinum toxin, which causesa neuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

[0021] Botulinum toxin type A is the most lethal natural biologicalagent known to man. About 50 picograms of a commercially availablebotulinum toxin type A (purified neurotoxin complex)¹ is a LD₅₀ in mice(i.e. 1 unit). One unit of BOTOX® contains about 50 picograms (about 56attomoles) of botulinum toxin type A complex. Interestingly, on a molarbasis, botulinum toxin type A is about 1.8 billion times more lethalthan diphtheria, about 600 million times more lethal than sodiumcyanide, about 30 million times more lethal than cobra toxin and about12 million times more lethal than cholera. Singh, Critical Aspects ofBacterial Protein Toxins, pages 63-84 (chapter 4) of Natural Toxins II,edited by B. R. Singh et al., Plenum Press, New York (1996) (where thestated LD₅₀ of botulinum toxin type A of 0.3 ng equals 1 U is correctedfor the fact that about 0.05 ng of BOTOX® equals 1 unit). One unit (U)of botulinum toxin is defined as the LD₅₀ upon intraperitoneal injectioninto female Swiss Webster mice weighing 18 to 20 grams each.

[0022] Seven immunologically distinct botulinum neurotoxins have beencharacterized, these being respectively botulinum neurotoxin serotypesA, B, C₁, D, E, F and G each of which is distinguished by neutralizationwith type-specific antibodies. The different serotypes of botulinumtoxin vary in the animal species that they affect and in the severityand duration of the paralysis they evoke. For example, it has beendetermined that botulinum toxin type A is 500 times more potent, asmeasured by the rate of paralysis produced in the rat, than is botulinumtoxin type B. Additionally, botulinum toxin type B has been determinedto be non-toxic in primates at a dose of 480 U/kg which is about 12times the primate LD₅₀ for botulinum toxin type A. Botulinum toxinapparently binds with high affinity to cholinergic motor neurons, istranslocated into the neuron and blocks the release of acetylcholine.

[0023] Regardless of serotype, the molecular mechanism of toxinintoxication appears to be similar and to involve at least three stepsor stages. In the first step of the process, the toxin binds to thepresynaptic membrane of the target neuron through a specific interactionbetween the heavy chain, H chain, and a cell surface receptor; thereceptor is thought to be different for each type of botulinum toxin andfor tetanus toxin. The carboxyl end segment of the H chain, H_(c),appears to be important for targeting of the toxin to the cell surface.

[0024] In the second step, the toxin crosses the plasma membrane of thepoisoned cell. The toxin is first engulfed by the cell throughreceptor-mediated endocytosis, and an endosome containing the toxin isformed. The toxin then escapes the endosome into the cytoplasm of thecell. This step is thought to be mediated by the amino end segment ofthe H chain, H_(N), which triggers a conformational change of the toxinin response to a pH of about 5.5 or lower. Endosomes are known topossess a proton pump which decreases intraendosomal pH. Theconformational shift exposes hydrophobic residues in the toxin, whichpermits the toxin to embed itself in the endosomal membrane. The toxin(or at a minimum the light chain) then translocates through theendosomal membrane into the cytoplasm.

[0025] The last step of the mechanism of botulinum toxin activityappears to involve reduction of the disulfide bond joining the heavychain, H chain, and the light chain, L chain. The entire toxic activityof botulinum and tetanus toxins is contained in the L chain of theholotoxin; the L chain is a zinc (Zn++) endopeptidase which selectivelycleaves proteins essential for recognition and docking ofneurotransmitter-containing vesicles with the cytoplasmic surface of theplasma membrane, and fusion of the vesicles with the plasma membrane.Tetanus neurotoxin, and botulinum toxins B, D, F, and G causedegradation of synaptobrevin (also called vesicle-associated membraneprotein (VAMP)), a synaptosomal membrane protein. Most of the VAMPpresent at the cytoplasmic surface of the synaptic vesicle is removed asa result of any one of these cleavage events. Serotype A and E cleaveSNAP-25. Serotype C₁ was originally thought to cleave syntaxin, but wasfound to cleave syntaxin and SNAP-25. Each toxin specifically cleaves adifferent bond (except tetanus and type B which cleave the same bond).

[0026] Botulinum toxins have been used in clinical settings for thetreatment of neuromuscular disorders characterized by hyperactiveskeletal muscles. Botulinum toxin type A was approved by the U.S. Foodand Drug Administration in 1989 for the treatment of blepharospasm,strabismus and hemifacial spasm. Non-type A botulinum toxin serotypesapparently have a lower potency and/or a shorter duration of activity ascompared to botulinum toxin type A. Clinical effects of peripheralintramuscular botulinum toxin type A are usually seen within one week ofinjection. The typical duration of symptomatic relief from a singleintramuscular injection of botulinum toxin type A averages about threemonths.

[0027] Although all the botulinum toxins serotypes apparently inhibitrelease of the neurotransmitter acetylcholine at the neuromuscularjunction, they do so by affecting different neurosecretory proteinsand/or cleaving these proteins at different sites. For example,botulinum types A and E both cleave the 25 kiloDalton (kD) synaptosomalassociated protein (SNAP-25), but they target different amino acidsequences within this protein Botulinum toxin types B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin type C₁ has been shown to cleave both syntaxin and SNAP-25. Thesedifferences in mechanism of action may affect the relative potencyand/or duration of action of the various botulinum toxin serotypes.Apparently, a substrate for a botulinum toxin can be found in a varietyof different cell types. See e.g. Biochem, J 1;339 (pt 1):159-65:1999,and Mov Disord, 10(3):376:1995 (pancreatic islet B cells contain atleast SNAP-25 and synaptobrevin).

[0028] The molecular weight of the botulinum toxin protein molecule, forall seven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ is apparentlyproduced as only a 700 kD or 500 kD complex. Botulinum toxin type D isproduced as both 300 kD and 500 kD complexes. Finally, botulinum toxintypes E and F are produced as only approximately 300 kD complexes. Thecomplexes (i.e. molecular weight greater than about 150 kD) are believedto contain a non-toxin hemaglutinin protein and a non-toxin andnon-toxic nonhemaglutinin protein. These two non-toxin proteins (whichalong with the botulinum toxin molecule comprise the relevant neurotoxincomplex) may act to provide stability against denaturation to thebotulinum toxin molecule and protection against digestive acids whentoxin is ingested. Additionally, it is possible that the larger (greaterthan about 150 kD molecular weight) botulinum toxin complexes may resultin a slower rate of diffusion of the botulinum toxin away from a site ofintramuscular injection of a botulinum toxin complex.

[0029] In vitro studies have indicated that botulinum toxin inhibitspotassium cation induced release of both acetylcholine andnorepinephrine from primary cell cultures of brainstem tissue.Additionally, it has been reported that botulinum toxin inhibits theevoked release of both glycine and glutamate in primary cultures ofspinal cord neurons and that in brain synaptosome preparations botulinumtoxin inhibits the release of each of the neurotransmittersacetylcholine, dopamine, norepinephrine (Habermann E., et al., TetanusToxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline ReleaseFrom Cultured Mouse Brain, J Neurochem 51(2);522-527:1988) CGRP,substance P and glutamate (Sanchez-Prieto, J., et al., Botulinum Toxin ABlocks Glutamate Exocytosis From Guinea Pig Cerebral CorticalSynaptosomes, Eur J. Biochem 165;675-681:1987. Thus, when adequateconcentrations are used, stimulus-evoked release of mostneurotransmitters is blocked by botulinum toxin. See e.g. Pearce, L. B.,Pharmacologic Characterization of Botulinum Toxin For Basic Science andMedicine, Toxicon 35(9);1373-1412 at 1393 (1997); Bigalke H., et al.,Botulinum A Neurotoxin Inhibits Non-Cholinergic Synaptic Transmission inMouse Spinal Cord Neurons in Culture, Brain Research 360;318-324:1985;Habermann E., Inhibition by Tetanus and Botulinum A Toxin of the Releaseof [ ³ H]Noradrenaline and [ ³ H]GABA From Rat Brain Homogenate,Experientia 44;224-226:1988, Bigalke H., et al., Tetanus Toxin andBotulinum A Toxin Inhibit Release and Uptake of Various Transmitters, asStudied with Particulate Preparations From Rat Brain and Spinal Cord,Naunyn-Schmiedeberg's Arch Pharmacol 316;244-251:1981, and; Jankovic J.et al., Therapy With Botulinum Toxin, Marcel Dekker, Inc., (1994), page5.

[0030] Botulinum toxin type A can be obtained by establishing andgrowing cultures of Clostridium botulinum in a fermenter and thenharvesting and purifying the fermented mixture in accordance with knownprocedures. All the botulinum toxin serotypes are initially synthesizedas inactive single chain proteins which must be cleaved or nicked byproteases to become neuroactive. The bacterial strains that makebotulinum toxin serotypes A and G possess endogenous proteases andserotypes A and G can therefore be recovered from bacterial cultures inpredominantly their active form. In contrast, botulinum toxin serotypesC₁, D and E are synthesized by nonproteolytic strains and are thereforetypically unactivated when recovered from culture. Serotypes B and F areproduced by both proteolytic and nonproteolytic strains and thereforecan be recovered in either the active or inactive form. However, eventhe proteolytic strains that produce, for example, the botulinum toxintype B serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

[0031] High quality crystalline botulinum toxin type A can be producedfrom the Hall A strain of Clostridium botulinum with characteristics of≧3×10⁷ U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern ofbanding on gel electrophoresis. The known Shantz process can be used toobtain crystalline botulinum toxin type A, as set forth in Shantz, E.J., et al, Properties and Use of Botulinum Toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56;80-99:1992. Generally, thebotulinum toxin type A complex can be isolated and purified from ananaerobic fermentation by cultivating Clostridium botulinum type A in asuitable medium. The known process can also be used, upon separation outof the non-toxin proteins, to obtain pure botulinum toxins, such as forexample: purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, and; purified botulinum toxin type F with an approximately 155kD molecular weight with a specific potency of 1-2×10⁷ LD₅₀ U/mg orgreater.

[0032] Botulinum toxins and/or botulinum toxin complexes can be obtainedfrom List Biological Laboratories, Inc., Campbell, Calif.; the Centrefor Applied Microbiology and Research, Porton Down, U. K.; Wako (Osaka,Japan), Metabiologics (Madison, Wis.) as well as from Sigma Chemicals ofSt. Louis, Mo.

[0033] Pure botulinum toxin is so labile that it is generally not usedto prepare a pharmaceutical composition. Furthermore, the botulinumtoxin complexes, such as the toxin type A complex are also extremelysusceptible to denaturation due to surface denaturation, heat, andalkaline conditions. Inactivated toxin forms toxoid proteins which maybe immunogenic. The resulting antibodies can render a patient refractoryto toxin injection.

[0034] As with enzymes generally, the biological activities of thebotulinum toxins (which are intracellular peptidases) are dependent, atleast in part, upon their three dimensional conformation. Thus,botulinum toxin type A is detoxified by heat, various chemicals surfacestretching and surface drying. Additionally, it is known that dilutionof the toxin complex obtained by the known culturing, fermentation andpurification to the much, much lower toxin concentrations used forpharmaceutical composition formulation results in rapid detoxificationof the toxin unless a suitable stabilizing agent is present. Dilution ofthe toxin from milligram quantities to a solution containing nanogramsper milliliter presents significant difficulties because of the rapidloss of specific toxicity upon such great dilution. Additionally, thetoxin may be used months or years after the toxin containingpharmaceutical composition is formulated. Significantly, it is knownthat the toxin can be stabilized during the manufacture and compoundingprocesses as well as during storage by use of a stabilizing agent suchas albumin and gelatin.

[0035] A commercially available botulinum toxin containingpharmaceutical composition is sold under the trademark BOTOX® (availablefrom Allergan, Inc., of Irvine, Calif.). BOTOX® consists of a purifiedbotulinum toxin type A complex, albumin and sodium chloride packaged insterile, vacuum-dried form. The botulinum toxin type A is made from aculture of the Hall strain of Clostridium botulinum grown in a mediumcontaining N-Z amine and yeast extract. The botulinum toxin type Acomplex is purified from the culture solution by a series of acidprecipitations to a crystalline complex consisting of the active highmolecular weight toxin protein and an associated hemagglutinin protein.The crystalline complex is re-dissolved in a solution containing salineand albumin and sterile filtered (0.2 microns) prior to vacuum-drying.The vacuum-dried product is stored in a freezer at or below 5° C. BOTOX®can be reconstituted with sterile, non-preserved saline prior tointramuscular injection. Each vial of BOTOX® contains about 100 units(U) of Clostridium botulinum toxin type A purified neurotoxin complex,0.5 milligrams of human serum albumin and 0.9 milligrams of sodiumchloride in a sterile, vacuum-dried form without a preservative.

[0036] To reconstitute vacuum-dried BOTOX®, sterile normal salinewithout a preservative (0.9% Sodium Chloride Injection) is used bydrawing up the proper amount of diluent in the appropriate size syringe.Since BOTOX® may be denatured by bubbling or similar violent agitation,the diluent is gently injected into the vial. For sterility reasonsBOTOX® is preferably administered within four hours after the vial isremoved from the freezer and reconstituted. During these four hours,reconstituted BOTOX® can be stored in a refrigerator at about 20° C. toabout 8° C. Reconstituted, refrigerated BOTOX® retains its potency forat least two weeks. Neurology, 48:249-53:1997.

[0037] It has been reported that botulinum toxin type A has been used inclinical settings as follows:

[0038] (1) about 75-125 units of BOTOX® per intramuscular injection(multiple muscles) to treat cervical dystonia;

[0039] (2) 5-10 units of BOTOX® per intramuscular injection to treatglabellar lines (brow furrows) (5 units injected intramuscularly intothe procerus muscle and 10 units injected intramuscularly into eachcorrugator supercilii muscle);

[0040] (3) about 30-80 units of BOTOX® to treat constipation byintrasphincter injection of the puborectalis muscle;

[0041] (4) about 1-5 units per muscle of intramuscularly injected BOTOX®to treat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

[0042] (5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

[0043] (6) to treat upper limb spasticity following stroke byintramuscular injections of BOTOX® into five different upper limb flexormuscles, as follows:

[0044] (a) flexor digitorum profundus: 7.5 U to 30 U

[0045] (b) flexor digitorum sublimus: 7.5 U to 30 U

[0046] (c) flexor carpi ulnaris: 10 U to 40 U

[0047] (d) flexor carpi radialis: 15 U to 60 U

[0048] (e) biceps brachii: 50 U to 200 U. Each of the five indicatedmuscles has been injected at the same treatment session, so that thepatient receives from 90 U to 360 U of upper limb flexor muscle BOTOX®by intramuscular injection at each treatment session.

[0049] (7) to treat migraine, pericranial injected (injectedsymmetrically into glabellar, frontalis and temporalis muscles)injection of 25 U of BOTOX® has showed significant benefit as aprophylactic treatment of migraine compared to vehicle as measured bydecreased measures of migraine frequency, maximal severity, associatedvomiting and acute medication use over the three month period followingthe 25 U injection.

[0050] It is known that botulinum toxin type A can have an efficacy forup to 12 months (European J. Neurology 6 (Supp 4):S111-S1150:1999), andin some circumstances for as long as 27 months, (The Laryngoscope 109:1344-1346:1999). However, the usual duration of an intramuscularinjection of Botox® is typically about 3 to 4 months.

[0051] The success of botulinum toxin type A to treat a variety ofclinical conditions has led to interest in other botulinum toxinserotypes. A study of two commercially available botulinum type Apreparations (BOTOX® and Dysport) and preparations of botulinum toxinstype B and F (both obtained from Wako Chemicals, Japan) has been carriedout to determine local muscle weakening efficacy, safety and antigenicpotential. Botulinum toxin preparations were injected into the head ofthe right gastrocnemius muscle (0.5 to 200.0 units/kg) and muscleweakness was assessed using the mouse digit abduction scoring assay(DAS). ED₅₀ values were calculated from dose response curves. Additionalmice were given intramuscular injections to determine LD₅₀ doses. Thetherapeutic index was calculated as LD₅₀/ED₅₀. Separate groups of micereceived hind limb injections of BOTOX® (5.0 to 10.0 units/kg) orbotulinum toxin type B (50.0 to 400.0 units/kg), and were tested formuscle weakness and increased water consumption, the later being aputative model for dry mouth. Antigenic potential was assessed bymonthly intramuscular injections in rabbits (1.5 or 6.5 ng/kg forbotulinum toxin type B or 0.15 ng/kg for BOTOX®). Peak muscle weaknessand duration were dose related for all serotypes. DAS ED₅₀ values(units/kg) were as follows: BOTOX®: 6.7, Dysport®: 24.7, botulinum toxintype B: 27.0 to 244.0, botulinum toxin type F: 4.3. BOTOX® had a longerduration of action than botulinum toxin type B or botulinum toxin typeF. Therapeutic index values were as follows: BOTOX®: 10.5, Dysport®:6.3, botulinum toxin type B: 3.2. Water consumption was greater in miceinjected with botulinum toxin type B than with BOTOX®, althoughbotulinum toxin type B was less effective at weakening muscles. Afterfour months of injections 2 of 4 (where treated with 1.5 ng/kg) and 4 of4 (where treated with 6.5 ng/kg) rabbits developed antibodies againstbotulinum toxin type B. In a separate study, 0 of 9 BOTOX® treatedrabbits demonstrated antibodies against botulinum toxin type A. DASresults indicate relative peak potencies of botulinum toxin type A beingequal to botulinum toxin type F, and botulinum toxin type F beinggreater than botulinum toxin type B. With regard to duration of effect,botulinum toxin type A was greater than botulinum toxin type B, andbotulinum toxin type B duration of effect was greater than botulinumtoxin type F. As shown by the therapeutic index values, the twocommercial preparations of botulinum toxin type A (BOTOX® and Dysport®)are different. The increased water consumption behavior observedfollowing hind limb injection of botulinum toxin type B indicates thatclinically significant amounts of this serotype entered the murinesystemic circulation. The results also indicate that in order to achieveefficacy comparable to botulinum toxin type A, it is necessary toincrease doses of the other serotypes examined. Increased dosage cancomprise safety. Furthermore, in rabbits, type B was more antigenic thanwas BOTOX®, possibly because of the higher protein load injected toachieve an effective dose of botulinum toxin type B. Eur J Neurol 1999Nov;6(Suppl 4):S3-S10.

[0052] In addition to having pharmacologic actions at a peripherallocation, a botulinum toxin can also exhibit a denervation effect in thecentral nervous system. Wiegand et al, Naunyn-Schmiedeberg's Arch.Pharmacol. 1976; 292, 161-165, and Habermann, Naunyn-Schmiedeberg'sArch. Pharmacol. 1974; 281, 47-56 reported that botulinum toxin is ableto ascend to the spinal area by retrograde transport. As such, abotulinum toxin injected at a peripheral location, for exampleintramuscularly, can potentially be retrograde transported to the spinalcord.

[0053] U.S. Pat. No. 5,989,545 discloses that a modified clostridialneurotoxin or fragment thereof, preferably a botulinum toxin, chemicallyconjugated or recombinantly fused to a particular targeting moiety canbe used to treat pain by administration of the agent to the spinal cord.

[0054] Acetylcholine

[0055] Typically only a single type of small molecule neurotransmitteris released by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic as most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephine. In most instances acetylcholine has an excitatory effect.However, acetylcholine is known to have inhibitory effects at some ofthe peripheral parasympathetic nerve endings, such as inhibition ofheart rate by the vagal nerve.

[0056] The efferent signals of the autonomic nervous system aretransmitted to the body through either the sympathetic nervous system orthe parasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Sincethe preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

[0057] Acetylcholine activates two types of receptors, muscarinic andnicotinic receptors. The muscarinic receptors are found in all effectorcells stimulated by the postganglionic, neurons of the parasympatheticnervous system as well as in those stimulated by the postganglioniccholinergic neurons of the sympathetic nervous system. The nicotinicreceptors are found in the adrenal medulla, as well as within theautonomic ganglia, that is on the cell surface of the postganglionicneuron at the synapse between the preganglionic and postganglionicneurons of both the sympathetic and parasympathetic systems. Nicotinicreceptors are also found in many nonautonomic nerve endings, for examplein the membranes of skeletal muscle fibers at the neuromuscularjunction.

[0058] Acetylcholine is released from cholinergic neurons when small,clear, intracellular vesicles fuse with the presynaptic neuronal cellmembrane. A wide variety of non-neuronal secretory cells, such as,adrenal medulla (as well as the PC12 cell line) and pancreatic isletcells release catecholamines and parathyroid hormone, respectively, fromlarge dense-core vesicles. The PC12 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,permeabilized (as by electroporation) or by direct injection of thetoxin into the denervated cell. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

[0059] A neuromuscular junction is formed in skeletal muscle by theproximity of axons to muscle cells. A signal transmitted through thenervous system results in an action potential at the terminal axon, withactivation of ion channels and resulting release of the neurotransmitteracetylcholine from intraneuronal synaptic vesicles, for example at themotor endplate of the neuromuscular junction. The acetylcholine crossesthe extracellular space to bind with acetylcholine receptor proteins onthe surface of the muscle end plate. Once sufficient binding hasoccurred, an action potential of the muscle cell causes specificmembrane ion channel changes, resulting in muscle cell contraction. Theacetylcholine is then released from the muscle cells and metabolized bycholinesterases in the extracellular space. The metabolites are recycledback into the terminal axon for reprocessing into further acetylcholine.

[0060] Therefore, a need exists for a biocompatible, nonimmunogenic,nonbiodegradable implant which permits long term continuous release of atherapeutically effective neurotoxin in a human patient.

SUMMARY

[0061] The present invention meets this need and provides abiocompatible, nonimmunogenic, nonbiodegradable implant which permitslong term, continuous release of a neurotoxin in a human patient.

[0062] Our invention provides a neurotoxin implant which overcomes theknown problems, difficulties and deficiencies associated with repetitivebolus or subcutaneous injection of a neurotoxin, such as a botulinumtoxin, to treat an affliction such as a movement disorder, including amuscle spasm.

[0063] A controlled release system within the scope of our inventioncomprises a polymeric matrix, and a quantity of neurotoxin locatedwithin the polymeric matrix, wherein fractional amounts of theneurotoxin can be released from the polymeric matrix over a prolongedperiod of time.

[0064] The neurotoxin can be released from the polymeric matrix in asubstantially continuous or monophasic manner and the prolonged periodof time during which neurotoxin is released from the polymeric matrixcan be from 10 days to about 6 years.

[0065] The polymeric matrix can be made of a substance which issubstantially non-biodegradable and the neurotoxin can be a polypeptide.Additionally, the neurotoxin can be a presynaptic neurotoxin, such as aClostridial neurotoxin. Further, the neurotoxin can be a botulinumtoxin, such as a botulinum toxin selected from the group consisting ofbotulinum toxin types A, B, C₁, D, E, F and G. Preferably, theneurotoxin is a botulinum toxin type A.

[0066] The polymer which comprises the polymeric matrix is selected fromthe group consisting of methacrylate, vinyl pyrrolidone, vinyl alcohol,acrylic acid, siloxane, vinyl acetate, lactic acid, glycolic acid,collagen, and bioceramic polymers and copolymers thereof.

[0067] The quantity of the neurotoxin held by the implant is betweenabout 1 unit and about 100,000 units of a botulinum toxin andpreferably, from about 1 to about 50,000 units of a botulinum toxin.Thus, the quantity of the neurotoxin can be between about 10 units andabout 2,000 units of a botulinum toxin type A and the quantity of theneurotoxin can be between about 100 units and about 30,000 units of abotulinum toxin type B.

[0068] The neurotoxin can be a botulinum toxin which is released fromthe implant in an amount effective to cause flaccid muscular paralysisof a muscle or muscle group at or in the vicinity of the implantedsystem.

[0069] A detailed embodiment of the present invention can be acontrolled release system comprising a polymeric matrix, and betweenabout 10 units and about 20,000 units of a botulinum toxin within thepolymeric matrix, wherein fractional amounts of the botulinum toxin canbe released from the polymeric matrix over a prolonged period of timeextending from about 2 months to about 5 years.

[0070] A method for making a controlled release system within the scopeof our invention can have the steps of (a) dissolving a polymer in asolvent to form a polymer solution; (b) mixing or dispersing aneurotoxin in the polymer solution to form a polymer-neurotoxin mixture,and; (c) allowing the polymerneurotoxin mixture to set, thereby making acontrolled release system. There can also be the step after the mixingstep of evaporating solvent.

[0071] Additionally, a method for using a continuous release systemwithin the scope of our invention can comprise injection or implantationof a controlled release system which includes a polymeric matrix,thereby treating a movement disorder or a disorder influenced bycholinergic innervation.

[0072] Finally, a method for forming a metal cation-complexed neurotoxincomprising the steps of (a) forming a solution containing a neurotoxin;(b) dispersing a multivalent metal cation component with the neurotoxinsolution under pH conditions suitable for complexing the multivalentmetal cation with the neurotoxin, thereby forming a metalcation-complexed neurotoxin suspension wherein the molar ratio of metalcation component to neurotoxin is between about 4:1 to about 100:1; and;(c) drying said suspension to form the metal cation-complexedneurotoxin.

[0073] The amount of a neurotoxin administered by a continuous releasesystem within the scope of the present invention during a given periodcan be between about 10⁻³ U/kg and about 35 U/kg for a botulinum toxintype A and up to about 200 U/kg for other botulinum toxins, such as abotulinum toxin type B. 35 U/kg or 200 U/kg is an upper limit because itapproaches a lethal dose of certain neurotoxins, such as botulinum toxintype A and botulinum toxin type B, respectively. Preferably, the amountof the neurotoxin administered by a continuous release system during agiven period is between about 10⁻² U/kg and about 25 U/kg. Morepreferably, the neurotoxin is administered in an amount of between about10⁻¹ U/kg and about 15 U/kg. Most preferably, the neurotoxin isadministered in an amount of between about 1 U/kg and about 10 U/kg. Inmany instances, an administration of from about 1 units to about 500units of a neurotoxin, such as a botulinum toxin type A, provideseffective and long lasting therapeutic relief. More preferably, fromabout 5 units to about 300 units of a neurotoxin, such as a botulinumtoxin type A, can be used and most preferably, from about 10 units toabout 200 units of a neurotoxin, such as a botulinum toxin type A, canbe locally administered into a target tissue with efficacious results.In a particularly preferred embodiment of the present invention fromabout 1 units to about 100 units of a botulinum toxin, such as botulinumtoxin type A, can be locally administered into a target tissue withtherapeutically effective results.

[0074] The neurotoxin can be made by a Clostridial bacterium, such as bya Clostridium botulinum, Clostridium butyricum, Clostridium beratti orClostridium tetani bacterium. Additionally, the neurotoxin can be amodified neurotoxin, that is a neurotoxin that has at least one of itsamino acids deleted, modified or replaced, as compared to the native orwild type neurotoxin. Furthermore, the neurotoxin can be a recombinantproduced neurotoxin or a derivative or fragment thereof.

[0075] The neurotoxin can be a botulinum toxin, such as one of thebotulinum toxin serotypes A, B, C₁, D, E, F or G. Preferably, theneurotoxin is botulinum toxin type A.

[0076] Significantly, the botulinum toxin can be is administered to bysubdermal implantation to the patient by placement of a botulinum toxinimplant. The botulinum toxin can administered to a muscle of a patientin an amount of between about 1 unit and about 10,000 units. When thebotulinum toxin is botulinum toxin type A and the botulinum toxin can beadministered to a muscle of the patient in an amount of between about 1unit and about 100 units.

[0077] Notably, it has been reported that glandular tissue treated by abotulinum toxin can show a reduced secretory activity for as long as 27months post injection of the toxin. Laryngoscope 1999; 109:1344-1346,Laryngoscope 1998;108:381-384.

[0078] Our invention relates to an implant for the controlled release ofa neurotoxin and to methods for making and using such implants. Theimplant can comprise a polymer matrix containing a neurotoxin. Theimplant is designed to administer effective levels of neurotoxin over aprolonged period of time when administered, for example,intramuscularly, epidurally or subcutaneously for the treatment ofvarious diseases conditions.

[0079] This invention further relates to a composition, and methods ofmaking and using the composition, for the controlled of biologicallyactive, stabilized neurotoxin. The controlled release composition ofthis invention can comprise a polymeric matrix of a biocompatiblepolymer and biologically active, stabilized neurotoxin dispersed withinthe biocompatible polymer.

[0080] Definitions

[0081] The following definitions apply herein.

[0082] “Biocompatible” means that there is an insignificant inflammatoryresponse at the site of implantation from use of the implant.

[0083] “Biologically active compound” means a compound which can effecta beneficial change in the subject to which it is administered. Forexample, “biologically active compounds” include neurotoxins.

[0084] “Effective amount” as applied to the biologically active compoundmeans that amount of the compound which is generally sufficient toeffect a desired change in the subject. For example, where the desiredeffect is a flaccid muscle paralysis, an effective amount of thecompound is that amount which causes at least a substantial paralysis ofthe desired muscles without causing a substantial paralysis of adjacentmuscle of which paralysis is not desired, and without resulting in asignificant systemic toxicity reaction.

[0085] “Effective amount” as applied to a non-active ingredientconstituent of an implant (such as a polymer used for forming a matrixor a coating composition) refers to that amount of the non-activeingredient constituent which is sufficient to positively influence therelease of a biologically active agent at a desired rate for a desiredperiod of time. For example, where the desired effect is muscleparalysis by using a single implant, the “effective amount” is theamount that can facilitate extending the release over a period ofbetween about 60 days and 6 years. This “effective amount” can bedetermined based on the teaching in this specification and the generalknowledge in the art.

[0086] “Effective amount” as applied to the amount of surface area of animplant is that amount of implant surface area which is sufficient toeffect a flux of biologically active compound so as to achieve a desiredeffect, such as a muscle paralysis. The area necessary may be determinedand adjusted directly by measuring the release obtained for theparticular active compound.

[0087] The surface area of the implant or of a coating of an implant isthat amount of membrane necessary to completely encapsulate thebiologically active compound. The surface area depends on the geometryof the implant. Preferably, the surface area is minimized wherepossible, to reduce the size of the implant.

[0088] “Implant” means a controlled release drug delivery system. Theimplant is comprised of a biocompatible polymer or ceramic materialwhich contains or which can act as a carrier for a molecule with abiological activity. The implant can be, injected, inserted or implantedinto a human body.

[0089] “Local administration” means direct administration of abiologically active compound, such as a therapeutic drug to a tissue bya non-systemic route. Local administration therefore includes,subcutaneous, intramuscular, intraspinal (i.e. intrathecal andepidural), intracranial, and intraglandular administration. Localadministration excludes a systemic route of administration such as oralor intravenous administration.

[0090] “Neurotoxin” means an agent which can interrupt nerve impulsetransmission across a neuromuscular or neuroglandular junction, block orreduce neuronal exocytosis of a neurotransmitter or alter the actionpotential at a sodium channel voltage gate of a neuron. Examples ofneurotoxins include botulinum toxins, tetanus toxins, saxitoxins, andtetrodotoxin.

[0091] “Treatment” means any treatment of a disease in a mammal, andincludes: (i) preventing the disease from occurring or; (ii) inhibitingthe disease, i.e., arresting its development; (iii) relieving thedisease, i.e., reducing the incidence of symptoms of or causingregression of the disease.

[0092] A method for making an implant within the scope of the presentinvention for controlled release of a neurotoxin, can include dissolvinga biocompatible polymer in a polymer solvent to form a polymer solution,dispersing particles of biologically active, stabilized neurotoxin inthe polymer solution, and then solidifying the polymer to form apolymeric matrix containing a dispersion of the neurotoxin particles.

[0093] A method of using an implant within the scope of the presentinvention forming for controlled release of a neurotoxin can compriseproviding a therapeutically effective level of biologically active,neurotoxin in a patient for a prolonged period of time by implanting inthe patient the implant.

DESCRIPTION

[0094] The present invention is based upon the discovery that acontinuous release, implant comprising a biocompatible,non-biodegradable or biodegradable polymer can exhibit prolonged in vivorelease of therapeutic amounts of a neurotoxin.

[0095] An implant within the scope of our invention can be surgicallyinserted by incision at the site of desired effect (i.e. for reductionof a muscle spasm) or the implant can be administered subcutaneously orintramuscularly using a hollow needle implanting gun, for example of thetype disclosed in U.S. Pat. No. 4,474,572. The diameter of the needlemay be adjusted to correspond to the size of the implant used. Further,an implant within the scope of the present invention can be implantedintracranially so as to provide long term delivery of a therapeuticamount of a neurotoxin to a target brain tissue. Removal of anon-biodegradable implant within the scope of the present invention isnot necessary once the implant has been spent, since the implant iscomprised of a biocompatible, nonimmunogenic material.

[0096] To stabilize a neurotoxin, both in a format which renders theneurotoxin useful for mixing with a suitable polymer which can form theimplant matrix (i.e. a powdered neurotoxin which has been freeze driedor lyophilized) as well as while the neurotoxin is present orincorporated into the matrix of the selected polymer, variouspharmaceutical excipients can be used. Suitable excipients can includestarch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride and dried skim milk.

[0097] The thickness of the implant can be used to control theabsorption of water by, and thus the rate of release of a neurotoxinfrom, a composition of the invention, thicker implants releasing thepolypeptide more slowly than thinner ones.

[0098] The implant can rapidly release a suboptimal amount of neurotoxinduring a first phase, the burst period. The burst period typically lastsless than 24 hours and frequently extends over only about an hour or soafter implantation. Thereafter the amount of neurotoxin released by theimplant rapidly declines and stabilizes at a much reduced and,significantly, relatively constant (i.e. zero order kinetics) level ofreleased neurotoxin. This second, prolonged phase of neurotoxin releasecan extend over a period of from about one year to about five or sixyears. An initial portion of the second phase can be termed the make upperiod.

[0099] The additive amount of neurotoxin released during the burst phaseand the make up period is preferably equal to an optimal amount ofneurotoxin so as to treat a particular disorder or affliction. Thetemporal extent of the make up period is somewhat less than the periodof time upon the expiry of which an optimal administration of theneurotoxin shows significantly reduced efficacy. For example, to treatupper limb spasticity the optimal amount of intramuscular botulinumtoxin type A can be about 90 units injected into the biceps brachiimuscle. Typically, the flaccid paralysis so induced within 1-7 days of abolus injection substantially wears off after about 3 months. Asubdermal neurotoxin implant within the scope of our invention can beconfigured to release about 60 units of botulinum toxin type essentiallyimmediately upon implantation (i.e. during the burst period). Thissuboptimal amount of neurotoxin provides rapid and substantial relief.During phase 2 the implant continuously releases about 0.4 unit/day of aneurotoxin, such as a botulinum toxin type A, so that after about 75days the optimal amount of 90 units has been released by the implantinto the target tissue.

[0100] The pre-synaptic neuronal receptor for which botulinum toxinexhibits a high and specific affinity has not been identified. Nor has agenerally accepted mechanism to account for the long intraneuronal halflife of botulinum toxin been elucidated. Nevertheless it is known that adynamic process, which may be either unblocking, reappearance,resynthesis and/or reactivation of the botulinum toxin receptor or theappearance of new neural sprouts, or both, transpires and accounts forthe gradual wearing off of the paralytic effect which results from anadministration of a botulinum toxin. Thus, while in the example above itcan take 75 days for an optimal amount (total of 90 units) of botulinumtoxin to be released by the implant, due to the dynamic nature of theattenuation of the effect of the botulinum toxin, subsequent release oftoxin (i.e. beyond 75 days) by the implant does not result in unwantedor excess areas of paralysis. Thus, it can be expected, in this example,that toxin released by the implant on day 76 binds to the new receptorsand/or neural sprouts formed in response to the denervation caused bythe toxin released by the implant on or about day one. The rollingnature of the denervation process means that, rather than resulting inexcess toxin which can diffuse systemically or cause unwanted paralysis,the continuous release of toxin after the end of the make up periodsimply again denervates within the same desired muscle location. Thus,assuming a spherical pattern of denervation and holding other factorsconstant, the burst release denervates a sphere of tissue with adiameter about ⅔ the optimal size of the tissue mass for whichdenervation is desired. Later release of neurotoxin during the make upperiod and subsequent provides the optimal or desired extent of tissuedenervation and amounts of neurotoxin to renew denervation at recentlyrenervated sites within the target tissue.

[0101] It is known that blepharospasm can be treated by intramuscularinjection of about 5 units (repeated at 2-4 month intervals) ofbotulinum toxin type A into the lateral pre-tarsal orbicularis oculimuscle. Significantly, a single implant within the scope of ourinvention can be used for the treatment of blepharospasm over, forexample, a one-year period. With this affliction and a one year periodchosen for treatment by implant release of neurotoxin, and a 15% burstcharacteristic polymer used, the total neurotoxin loading into theimplant can be 20 units. During the burst period about 3 units of thetoxin is released (within 24 hours after implantation) followed by acontinuous release of about 0.0467 units per day (i.e. about 2.3picograms of BOTOX® released per day). Thus, by day 42 about 5 unitstotal of the neurotoxin has been released. The release rate in thisexample (15% burst, remaining 85% over 364 days) is 0.234%/day. In thisexample, on day 1 the patient receives a 20 unit implant and one yearlater the patient has the spent implant removed and another 20 unitimplant inserted. Thus 25 units are administered over 365 days, witheffect of second implant on day 365 included.

[0102] Since one mole (M) of the botulinum toxin type A complex containsabout 9×10⁵ grams, therefore one picogram (pg) of the botulinum toxintype A complex is about 1.1×10⁻¹⁸ M. Hence, a desired release of0.234%/day of total incorporated neurotoxin equals a release of about2.53×10⁻¹⁸ M/day. With one year treatment period, 20% burst, followed by80% over 364 days results in a controlled release of about 0.22%/day or0.044 units/day or 2.2 picograms/day or about 2.42×10⁻¹⁸ M/day. A 20%burst from a 20 unit implant provides 4 units of the neurotoxin in aboutthe first 24 hours after implantation. Generally, surface area of theimplant is equal to x units of toxin released/day for each y cm² ofimplant surface area.

[0103] Different conditions are treated with botulinum toxin injectionranging from about 5 units to about 100 units per injection. A typicalimplant to treat, over a one year period, a condition for which 25 unitsof type A is the optimal bolus dose can be loaded with 100 units of abotulinum toxin type A complex. The burst can be 20%, followed by 80%over 364 days, which is equal to 0.22%/day or 0.22 units/day or 11picograms/day or about 1.21×10⁻¹⁷ M/day

[0104] For a five year treatment period, that is 20 bolus injections of25 units, the first injection is at time zero, and the 20^(th) injectionis at month 57, for a 500 unit total series of injections. Contrarily,with our invention, a 5 year implant to treat a condition responsive to25 units of a botulinum toxin, such as a botulinum toxin type A, can bemade with a 500 toxin unit loaded implant with the characteristics of a20 unit burst (4% burst), followed by about 480 units released overabout 1736 days, which is equal to 0.267 unit/day or 5.56×10⁻⁴%/day or13.35 picograms/day released by the implant.

[0105] A matrix implant can be made by dissolving a selected polymer inan appropriate solvent. Into this casting solution the desired amount oflyophilized or freeze dried, powered neurotoxin (i.e. the total desiredamount of the neurotoxin, such as non-reconstituted BOTOX®, to bereleased over the therapeutic period) is mixed. This method can be usedto make coated implant pellets, with the modification that the coatingused in an embodiment of the present invention is a bioerodible polymerwhich is impermeable to the neurotoxin. Thus, the neurotoxin does notdiffuse out of the matrix into the surrounding tissue until the coatinghas degraded.

[0106] The pH of the casting or other solution in which the botulinumtoxin is to be mixed is maintained at pH 4.2-6.8, because at pH above 7the stabilizing nontoxin proteins dissociate from the botulinum toxinresulting in gradual loss of toxicity. Preferably, the pH is betweenabout 5-6. Furthermore the temperature of the mixture/solution shouldnot exceed about 35 degrees Celsius, because the toxin is readilydetoxified when in a solution/mixture heated above about 40 degreesCelsius.

[0107] Suitable implants within the scope of the present invention forthe controlled in vivo release of a neurotoxin, such as a botulinumtoxin, can be prepared so that the implant releases the neurotoxin ineither a continuous or in a pulsatile fashion. “Continuous release”means release of toxin in a substantially monophasic manner, after theinitial burst phase. A continuous release can have a point ofinflection, but not a plateau phase. Continuous release does not requirea release from the implant of a similar amount of a neurotoxin per unitif time. A pulsatile release implant can release a neurotoxin is abiphasic or multiphase manner. Thus, a pulsatile release implant canhave a relatively short initial induction (burst) period, followed byperiods during which little or no neurotoxin is released.

[0108] A controlled release of biologically active neurotoxin is arelease which results in therapeutically effective, with negligibleserum levels, of biologically active, neurotoxin over a period longerthan that obtained following direct administration of aqueousneurotoxin. It is preferred that a controlled release be a release ofneurotoxin for a period of about six months or more, and more preferablyfor a period of about one year or more.

[0109] Suitable implants within the scope of the present invention forthe controlled in vivo release of a neurotoxin, such as a botulinumtoxin, can exhibit a continuous release or a pulsatile release of theneurotoxin. Additionally, the implant can comprise a non-biodegradableor a biodegradable polymeric material. Significantly, our inventionencompasses: (1) continuous release, nonbiodegradable neurotoxinimplants; (2) continuous release, biodegradable neurotoxin implants; (3)pulsatile release, nonbiodegradable neurotoxin implants, and; (4)pulsatile release, biodegradable implants, and each of these four typesof encompasses implant can be formulated into a variety ofconformations, suitable for sub-dermal injection or implantation such aspellets, discs, microspheres, films, rods and tubes, each of which canhave, for example, one or more coatings over a reservoir or matrixstructure.

[0110] An implant within the scope of our invention can also beformulated as a suspension for injection. Such suspensions may bemanufactured by general techniques well known in the pharmaceutical art,for example by milling the polylactide/polypeptide mixture in anultracentrifuge mill fitted with a suitable mesh screen, for example a120 mesh, and suspending the milled, screened particles in a solvent forinjection, for example propylene glycol, water optionally with aconventional viscosity increasing or suspending agent, oils or otherknown, suitable liquid vehicles for injection.

[0111] Denaturation of the encapsulated neurotoxin in the body at 37degrees C. for a prolonged period of time can be reduced by stabilizingthe neurotoxin by lyophilizing it with albumin, lyophilizing from anacidic solution, lyophilizing from a low moisture content solution(these three criteria can be met with regard to a botulinum toxin type Aby use of non-reconstituted Botox®) and using a specific polymer matrixcomposition.

[0112] Preferably, the release of biologically active neurotoxin in vivodoes not result in a significant immune system response during therelease period of the neurotoxin.

[0113] Matrix Stabilized Neurotoxin

[0114] We have discovered that a stabilized neurotoxin can comprisebiologically active, non-aggregated neurotoxin complexed with at leastone type of multivalent metal cation which has a valiancy of +2 or more.

[0115] Suitable multivalent metal cations include metal cationscontained in biocompatible metal cation components. A metal cationcomponent is biocompatible if the cation component is non-toxic to therecipient, in the quantities used, and also presents no significantdeleterious or untoward effects on the recipient's body, such as animmunological reaction at the injection site.

[0116] Preferably, the molar ratio of metal cation component toneurotoxin, for the metal cation stabilizing the neurotoxin, is betweenabout 4:1 to about 100:1 and more typically about 4:1 to about 10:1.

[0117] A preferred metal cation used to stabilize neurotoxin is Zn⁺⁺.Divalent zinc cations are preferred because botulinum toxin is known tobe a divalent zinc endopeptidase. In a more preferred embodiment, themolar ratio of metal cation component, containing Zn⁺⁺ cations, toneurotoxin is about 6:1.

[0118] The suitability of a metal cation for stabilizing neurotoxin canbe determined by one of ordinary skill in the art by performing avariety of stability indicating techniques such as polyacrylamide gelelectrophoresis, isoelectric focusing, reverse phase chromatography,HPLC and potency tests on neurotoxin lyophilized particles containingmetal cations to determine the potency of the neurotoxin afterlyophilization and for the duration of release from microparticles. Instabilized neurotoxin, the tendency of neurotoxin to aggregate within amicroparticle during hydration in vivo and/or to lose biologicalactivity or potency due to hydration or due to the process of forming acontrolled release composition, or due to the chemical characteristicsof a controlled release composition, is reduced by complexing at leastone type of metal cation with neurotoxin prior to contacting theneurotoxin with a polymer solution.

[0119] By our invention, stabilized neurotoxin is stabilized againstsignificant aggregation in vivo over the controlled release period.Significant aggregation is defined as an amount of aggregation resultingin aggregation of about 15% or more of the polymer encapsulated orpolymer matrix incorporated neurotoxin. Preferably, aggregation ismaintained below about 5% of the neurotoxin. More preferably,aggregation is maintained below about 2% of the neurotoxin present inthe polymer.

[0120] The neurotoxin in a neurotoxin controlled release composition canalso be mixed with other excipients, such as bulking agents oradditional stabilizing agents, such as buffers to stabilize theneurotoxin during lyophilization.

[0121] Bulking agents typically comprise inert materials. Suitablebulking agents are known to those skilled in the art.

[0122] A polymer, or polymeric matrix, suitable for the controlledrelease composition of the present invention, must be biocompatible. Apolymer is biocompatible if the polymer, and any degradation products ofthe polymer, are non-toxic to the recipient and also present nosignificant deleterious or untoward effects on the recipient's body,such as an immunological reaction at the injection site.

[0123] The polymer of the neurotoxin controlled release composition canbe made of a biodegradable material. Biodegradable, as defined herein,means the composition will degrade or erode in vivo to form smallerchemical species. Degradation can result, for example, by enzymatic,chemical and physical processes.

[0124] Suitable biocompatible, biodegradable polymers include, forexample, poly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, polycaprolactone, polycarbonates, polyesteramides,polyanhydrides, poly(amino acids), polyorthoesters, polycyanoacrylates,poly(p-dioxanone), poly(alkylene oxalates), biodegradable polyurethanes,blends and copolymers thereof.

[0125] Further, the terminal functionalities of the polymer can bemodified. For example, polyesters can be blocked, unblocked or a blendof blocked and unblocked polymers. A blocked polymer is as classicallydefined in the art, specifically having blocked carboxyl end groups.Generally, the blocking group is derived from the initiator of thepolymerization and is typically an alkyl group. An unblocked polymergenerally has free carboxyl end groups.

[0126] Acceptable molecular weights for a biodegradable polymer used inthis invention can be determined by a person of ordinary skill in theart taking into lo consideration factors such as the desired polymerdegradation rate, physical properties such as mechanical strength, andrate of dissolution of polymer in solvent. Typically, an acceptablerange of molecular weights is of about 2,000 Daltons to about 2,000,000Daltons. In a preferred embodiment, the polymer is a biodegradablepolymer or copolymer. In a more preferred A s embodiment, the polymer isa poly(lactide-co-glycolide) (hereinafter “PLGA”) with alactide:glycolide ratio of about 1:1 and a molecular weight of about5,000 Daltons to about 70,000 Daltons. In an even more preferredembodiment, the molecular weight of the PLGA used in the presentinvention has a molecular weight of about 6,000 to about 31,000 Daltons.

[0127] The amount of neurotoxin, which is contained in a dose ofcontrolled release microparticles, or in an alternate controlled releasesystem, containing biologically active, stabilized neurotoxin particlesis a therapeutically or prophylactically effective amount, which can bedetermined by a person of ordinary skill in the art taking intoconsideration factors such as body weight, condition to be treated, typeof polymer used, and release rate from the polymer.

[0128] In one embodiment, a neurotoxin controlled release compositioncontains from about 10⁻⁴% (w/w) to about 1% (w/w) of biologicallyactive, stabilized neurotoxin. The amount of such neurotoxin particlesused will vary depending upon the desired effect of the neurotoxin, theplanned release levels, the times at which neurotoxin should bereleased, and the time span over which the neurotoxin will be released.A preferred range of neurotoxin particle loading is between about 10⁻⁴%(w/w) to about 0.1% (w/w) neurotoxin particles. A more preferred rangeof neurotoxin loading is between about 10⁻³% (w/w) to about 1% (w/w)neurotoxin. The most preferred loading of the biologically active,stabilized neurotoxin particles is about 10⁻²% (w/w).

[0129] In another embodiment, a neurotoxin controlled releasecomposition also contains a second metal cation component, which is notcontained in the stabilized neurotoxin particles, and which is dispersedwithin the polymer. The second metal cation component preferablycontains the same species of metal cation, as is contained in thestabilized neurotoxin. Alternately, the second metal cation componentcan contain one or more different species of metal cation.

[0130] The second metal cation component acts to modulate the release ofthe neurotoxin from the polymeric matrix of the controlled releasecomposition, such as by acting as a reservoir of metal cations tofurther lengthen the period of time over which the neurotoxin isstabilized by a metal cation to enhance the stability of neurotoxin inthe composition.

[0131] A metal cation component used in modulating release typicallycontains at least one type of multivalent metal cation. Examples ofsecond metal cation components suitable to modulate neurotoxin release,include, or contain, for instance, Mg(OH)₂, MgCO₃ (such as4MgCO₃Mg(OH)₂5H₂O), ZnCO₃(such as 3Zn(OH)₂2ZnCO₃), CaCO₃, Zn₃ (C₆H₅O₇)₂,Mg(OAc)₂, MgSO₄, Zn(OAc)₂, ZnSO₄, ZnCl₂, MgCl₂ and Mg₃ (C₆H₅O₇)₂. Asuitable ratio of second metal cation component-to-polymer is betweenabout 1:99 to about 1:2 by weight. The optimum ratio depends upon thepolymer and the second metal cation component utilized.

[0132] The neurotoxin controlled release composition of this inventioncan be formed into many shapes such as a film, a pellet, a cylinder, adisc or a microparticle. A microparticle, as defined herein, comprises apolymeric component having a diameter of less than about one millimeterand having stabilized neurotoxin particles dispersed therein. Amicroparticle can have a spherical, non-spherical or irregular shape. Itis preferred that a microparticle be a microsphere. Typically, themicroparticle will be of a size suitable for injection. A preferred sizerange for microparticles is from about 1 to about 180 microns indiameter.

[0133] In the method of this invention for forming a composition for thecontrolled release of biologically active, non-aggregated neurotoxin, asuitable amount of particles of biologically active, stabilizedneurotoxin are dispersed in a polymer solution.

[0134] A suitable polymer solution contains between about 1% (w/w) andabout 30% (w/w) of a suitable biocompatible polymer, wherein thebiocompatible polymer is typically dissolved in a suitable polymersolvent. Preferably, a polymer solution contains about 2% (w/v) to about20% (w/v) polymer. A polymer solution containing 5% to about 10% (w/w)polymer is most preferred.

[0135] A suitable polymer solvent, as defined herein, is solvent inwhich the polymer is soluble but in which the stabilized neurotoxinparticles are substantially insoluble and non-reactive. Examples ofsuitable polymer solvents include polar organic liquids, such asmethylene chloride, chloroform, ethyl acetate and acetone.

[0136] To prepare biologically active, stabilized neurotoxin, neurotoxinis mixed in a suitable aqueous solvent with at least one suitable metalcation component under pH conditions suitable for forming a complex ofmetal cation and neurotoxin. Typically, the complexed neurotoxin will bein the form of a cloudy precipitate, which is suspended in the solvent.However, the complexed neurotoxin can also be in solution. In an evenmore preferred embodiment, neurotoxin is complexed with Zn⁺⁺.

[0137] Suitable pH conditions to form a complex of neurotoxin typicallyinclude pH values between about 5.0 and about 6.9. Suitable pHconditions are typically achieved through use of an aqueous buffer, suchas sodium bicarbonate, as the solvent.

[0138] Suitable solvents are those in which the neurotoxin and the metalcation component are each at least slightly soluble, such as in anaqueous sodium bicarbonate buffer. For aqueous solvents, it is preferredthat water used be either deionized water or water-for-injection (WFI).

[0139] The neurotoxin can be in a solid or a dissolved state, prior tobeing contacted with the metal cation component. Additionally, the metalcation component can be in a solid or a dissolved state, prior to beingcontacted with the neurotoxin. In a preferred embodiment, a bufferedaqueous solution of neurotoxin is mixed with an aqueous solution of themetal cation component.

[0140] Typically, the complexed neurotoxin will be in the form of acloudy precipitate, which is suspended in the solvent. However, thecomplexed neurotoxin can also be in solution. In a preferred embodiment,the neurotoxin is complexed with Zn⁺⁺.

[0141] The Zn⁺⁺ complexed neurotoxin can then be dried, such as bylyophilization, to form particulates of stabilized neurotoxin. The Zn⁺⁺complexed neurotoxin, which is suspended or in solution, can be bulklyophilized or can be divided into smaller volumes which are thenlyophilized. In a preferred embodiment, the Zn⁺⁺ complexed neurotoxinsuspension is micronized, such as by use of an ultrasonic nozzle, andthen lyophilized to form stabilized neurotoxin particles. Acceptablemeans to lyophilize the Zn⁺⁺ complexed neurotoxin mixture include thoseknown in the art.

[0142] Preferably, particles of stabilized neurotoxin are between about1 to about 6 micrometers in diameter. The neurotoxin particles can befragmented separately, Alternately, the neurotoxin particles can befragmented after being added to a polymer solution, such as by means ofan ultrasonic probe or ultrasonic nozzle.

[0143] In another embodiment, a second metal cation component, which isnot contained in the stabilized neurotoxin particles, is also dispersedwithin the polymer solution.

[0144] It is understood that a second metal cation component andstabilized neurotoxin can be dispersed into a polymer solutionsequentially, in reverse order, intermittently, separately or throughconcurrent additions. Alternately, a polymer, a second metal cationcomponent and stabilized neurotoxin and can be mixed into a polymersolvent sequentially, in reverse order, intermittently, separately orthrough concurrent additions.

[0145] In this method, the polymer solvent is then solidified to form apolymeric matrix containing a dispersion of stabilized neurotoxinparticles.

[0146] A suitable method for forming an neurotoxin controlled releasecomposition from a polymer solution is the solvent evaporation method isdescribed in U.S. Pat. Nos. 3,737,337; 3,523,906; 3,691,090, and;4,389,330. Solvent evaporation can be used as a method to formneurotoxin controlled release microparticles.

[0147] In the solvent evaporation method, a polymer solution containinga stabilized neurotoxin particle dispersion, is mixed in or agitatedwith a continuous phase, in which the polymer solvent is partiallymiscible, to form an emulsion. The continuous phase is usually anaqueous solvent. Emulsifiers are often included in the continuous phaseto stabilize the emulsion. The polymer solvent is then evaporated over aperiod of several hours or more, thereby solidifying the polymer to forma polymeric matrix having a dispersion of stabilized neurotoxinparticles contained therein.

[0148] A preferred method for forming neurotoxin controlled releasemicroparticles from a polymer solution is described in U.S. Pat. No.5,019,400. This method of microsphere formation, as compared to othermethods, such as phase separation, additionally reduces the amount ofneurotoxin required to produce a controlled release composition with aspecific neurotoxin content.

[0149] In this method, the polymer solution, containing the stabilizedneurotoxin particle dispersion, is processed to create droplets, whereinat least a significant portion of the droplets contain polymer solutionand the stabilized neurotoxin particles. These droplets are then frozenby means suitable to form microparticles. Examples of means forprocessing the polymer solution dispersion to form droplets includedirecting the dispersion through an ultrasonic nozzle, pressure nozzle,Rayleigh jet, or by other known means for creating droplets from asolution.

[0150] Means suitable for freezing droplets to form microparticlesinclude directing the droplets into or near a liquefied gas, such asliquid argon and liquid nitrogen to form frozen microdroplets which arethen separated from the liquid gas. The frozen microdroplets are thenexposed to a liquid non-solvent, such as ethanol, or ethanol mixed withhexane or pentane.

[0151] The solvent in the frozen microdroplets is extracted as a solidand/or liquid into the non-solvent to form stabilized neurotoxincontaining microparticles. Mixing ethanol with other non-solvents, suchas hexane or pentane, can increase the rate of solvent extraction, abovethat achieved by ethanol alone, from certain polymers, such aspoly(lactide-co-glycolide) polymers.

[0152] A wide range of sizes of neurotoxin controlled releasemicroparticles can be made by varying the droplet size, for example, bychanging the ultrasonic nozzle diameter. If very large microparticlesare desired, the microparticles can be extruded through a syringedirectly into the cold liquid. Increasing the viscosity of the polymersolution can also increase microparticle size. The size of themicroparticles can be produced by this process, for examplemicroparticles ranging from greater than about 1000 to about 1micrometers in diameter.

[0153] Yet another method of forming a neurotoxin controlled releasecomposition, from a polymer solution, includes film casting, such as ina mold, to form a film or a shape. For instance, after putting thepolymer solution containing a dispersion of stabilized neurotoxinparticles into a mold, the polymer solvent is then removed by meansknown in the art, or the temperature of the polymer solution is reduced,until a film or shape, with a consistent dry weight, is obtained.

[0154] In the case of a biodegradable polymer implant, release ofneurotoxin due to degradation of the polymer. The rate of degradationcan be controlled by changing polymer properties that influence the rateof hydration of the polymer. These properties include, for instance, theratio of different monomers, such as lactide and glycolide, comprising apolymer; the use of the L-isomer of a monomer instead of a racemicmixture; and the molecular weight of the polymer. These properties canaffect hydrophilicity and crystallinity, which control the rate ofhydration of the polymer. Hydrophilic excipients such as salts,carbohydrates and surfactants can also be incorporated to increasehydration and which can alter the rate of erosion of the polymer.

[0155] By altering the properties of a biodegradable polymer, thecontributions of diffusion and/or polymer degradation to neurotoxinrelease can be controlled. For example, increasing the glycolide contentof a poly(lactide-co-glycolide) polymer and decreasing the molecularweight of the polymer can enhance the hydrolysis of the polymer andthus, provides an increased neurotoxin release from polymer erosion. Inaddition, the rate of polymer hydrolysis is increased in non-neutralpH's. Therefore, an acidic or a basic excipient can be added to thepolymer solution, used to form the microsphere, to alter the polymererosion rate.

[0156] The composition of our invention can be administered to a human,or other animal, by any non-system means of administration, such as byimplantation (e.g. subcutaneously, intramuscularly, intracranially,intravaginally and intradermally), to provide the desired dosage ofneurotoxin based on the known parameters for treatment with neurotoxinof various medical conditions.

[0157] The specific dosage by implant appropriate for administration isreadily determined by one of ordinary skill in the art according to thefactor discussed above. The dosage can also depend upon the size of thetissue mass to be treated or denervated, and the commercial preparationof the toxin. Additionally, the estimates for appropriate dosages inhumans can be extrapolated from determinations of the amounts ofbotulinum required for effective denervation of other tissues. Thus, theamount of botulinum A to be injected is proportional to the mass andlevel of activity of the tissue to be treated. Generally, between about0.01 units per kilogram to about 35 units per kg of patient weight of abotulinum toxin, such as botulinum toxin type A, can be released by thepresent implant per unit time period (i.e. over a period of or onceevery 2-4 months) to effectively accomplish a desired muscle paralysis.Less than about 0.01 U/kg of a botulinum toxin does not have asignificant therapeutic effect upon a muscle, while more than about 35U/kg of a botulinum toxin approaches a toxic dose of a neurotoxin, suchas a botulinum toxin type A. Careful preparation and placement of theimplant prevents significant amounts of a botulinum toxin from appearingsystemically. A more preferred dose range is from about 0.01 U/kg toabout 25 U/kg of a botulinum toxin, such as that formulated as BOTOX®.The actual amount of U/kg of a botulinum toxin to be administereddepends upon factors such as the extent (mass) and level of activity ofthe tissue to be treated and the administration route chosen. Botulinumtoxin type A is a preferred botulinum toxin serotype for use in themethods of the present invention.

[0158] Preferably, a neurotoxin used to practice a method within thescope of the present invention is a botulinum toxin, such as one of theserotype A, B, C, D, E, F or G botulinum toxins. Preferably, thebotulinum toxin used is botulinum toxin type A, because of its highpotency in humans, ready availability, and known safe and efficacioususe for the treatment of skeletal muscle and smooth muscle disorderswhen locally administered by intramuscular injection.

[0159] The present invention includes within its scope the use of anyneurotoxin which has a long duration therapeutic effect when used totreat a movement disorder or an affliction influenced by cholinergicinnervation. For example, neurotoxins made by any of the species of thetoxin producing Clostridium bacteria, such as Clostridium botulinum,Clostridium butyricum, and Clostridium beratti can be used or adaptedfor use in the methods of the present invention. Additionally, all ofthe botulinum serotypes A, B, C, D, E, F and G can be advantageouslyused in the practice of the present invention, although type A is themost preferred serotype, as explained above. Practice of the presentinvention can provide effective relief for from 1 month to about 5 or 6years.

[0160] The present invention includes within its scope: (a) neurotoxincomplex as well as pure neurotoxin obtained or processed by bacterialculturing, toxin extraction, concentration, preservation, freeze dryingand/or reconstitution and; (b) modified or recombinant neurotoxin, thatis neurotoxin that has had one or more amino acids or amino acidsequences deliberately deleted, modified or replaced by knownchemical/biochemical amino acid modification procedures or by use ofknown host cell/recombinant vector recombinant technologies, as well asderivatives or fragments of neurotoxins so made, and includesneurotoxins with one or more attached targeting moieties for a cellsurface receptor present on a cell.

[0161] Botulinum toxins for use according to the present invention canbe stored in lyophilized or vacuum dried form in containers under vacuumpressure. Prior to lyophilization the botulinum toxin can be combinedwith pharmaceutically acceptable excipients, stabilizers and/orcarriers, such as albumin. The lyophilized or vacuum dried material canbe reconstituted with saline or water.

[0162] Our invention also includes within its scope the use of animplanted controlled release neurotoxin complex so as to providetherapeutic relief from a chronic disorder such as movement disorder.Thus, the neurotoxin can be imbedded within, absorbed, or carried by asuitable polymer matrix which can be implanted or embedded subdermallyso as to provide a year or more of delayed and controlled release of theneurotoxin to the desired target tissue. Implantable polymers whichpermit controlled release of polypeptide drugs are known, and can beused to prepare a botulinum toxin implant suitable for insertion orsubdermal attachment. See e.g. Pain 1999;82(1):49-55; Biomaterials1994;15(5):383-9; Brain Res 1990;515(1-2):309-11 and U.S. Pat. Nos.6,022,554; 6,011,011; 6,007,843; 5,667,808, and; 5,980,945.

[0163] Methods for determining the appropriate route of administrationand dosage are generally determined on a case by case basis by theattending physician. Such determinations are routine to one of ordinaryskill in the art (see for example, Harrison's Principles of InternalMedicine (1998), edited by Anthony Fauci et al., 14^(th) edition,published by McGraw Hill).

EXAMPLES

[0164] The following examples set forth specific compositions andmethods encompassed by the present invention and are not intended tolimit the scope of our invention.

Example 1 Formation of Zinc⁺⁺ Stabilized Neurotoxin

[0165] One hundred units of a neurotoxin, such as unreconstitutedBotox®, is dissolved in sodium bicarbonate buffer (pH 6.0) to form aneurotoxin solution. A Zn⁺⁺ solution is prepared from deionized waterand zinc acetate dihydrate and then added with gentle mixing to theneurotoxin solution to form a Zn⁺⁺ neurotoxin complex. The pH of theZn⁺⁺ neurotoxin complex is then adjusted to between 6.5 and 6.9 byadding 1% acetic acid. A cloudy suspended precipitate, comprisinginsoluble Zn⁺⁺ stabilized neurotoxin is thereby formed. There is therebymade a neurotoxin (such as a botulinum toxin type A) complex stabilizedagainst significant aggregation upon subsequent incorporation into apolymeric implant matrix.

Example 2 Neurotoxin Controlled Release Pellet

[0166] A neurotoxin suitable for incorporation into a polymer orpolymerizable solution can be a botulinum toxin type A (such as Botox®),which is commercially available as a freeze dried powder. Additionally,various polymers and copolymers can be mixed and stored in a dry statewith no effect on final implant performance. For example, an acrylatecopolymer using an UV cured initiator. The neurotoxin can be complexedwith Zn⁺⁺ as set forth in Example 1 above. The Zn⁺⁺ stabilizedneurotoxin complex is then mixed with uncured acrylate copolymer, UVinitiator and an acid (pH between 5.5 and 6.8). The mixture is placedinto a glass or clear plastic pellet mold which allows penetration of UVlight. The mold is placed into a temperature controlled water bath heldat 20° C. The pellet is cured with UV light for approximately 50seconds, packaged and sterilized. The duration and intensity of the UVcuring are such that insignificant amount of neurotoxin are disrupted ordenatured.

[0167] The size of the pellet and the concentration of the amount ofneurotoxin inside of the pellet are defined by the desired application.When the pellet is implanted, the pellet is hydrated inside of the body,which slightly delays the initial burst of the neurotoxin from inside ofthe implant. Coating the outside of the pellet with a portion of thedesired initial burst concentration of neurotoxin can offset this delay.In this example the pellet effectiveness would be for approximatelyabout 4 to about 6 months.

Example 3 Neurotoxin Controlled Release Formulations

[0168] To increase the amount of time the pellet can effectively deliverneurotoxin, multiple layers of materials can be used. Thus, the innermaterial can be made from a polyvinylpyrrolidone/methylmethacrylatecopolymer. This material allows for sustaining a high concentration ofneurotoxin complex. A suitable amount of neurotoxin is complexed withZn⁺⁺ as set forth in Example 1 above and this complex is then mixed withuncured copolymer, low temperature initiator and an acid (pH between 5.5and 6.8). The mixture is placed into a glass or plastic pellet mold. Themold is placed into a temperature controlled water bath at about 35degrees C. for between about 6 hours and about 8 hours. This forms thereservoir of neurotoxin required for a prolonged, controlled release.

[0169] In order to prolong the release of the neurotoxin a secondmaterial is then cured around the initial pellet. This material ischosen for high molecular density and biocompatibility.Polymethylmethacrylate (PMMA) is an example of a material with thischaracteristic. The pellet (above) is placed into a mold (insertionmolding) with uncured PMMA/low temperature initiator. A secondarycoating of the uncured PMMA maybe necessary to assure uniform coating ofthe pellet. Preferably, the PMMA thickness is 0.5 mm. After forming, theoutside of the pellet is coated with the desired initial burstconcentration of neurotoxin. The PMMA layer will be sufficiently thickto allow for a delay (up to 3 months) of the neurotoxin in thereservoir. When the neurotoxin reaches the surface of the implant asecond large burst of neurotoxin is obtained. This secondary burst willthen be followed by a slowly decreasing release rate of the neurotoxinfor approximately 3 months. In this example the pellet effectiveness isfor about 7 to about 9 months.

Example 4 Multi Layer Neurotoxin Controlled Release Implant

[0170] By utilizing multiple layers—high density polymer/low densitypolymer w/neurotoxin—the temporal extent of the controlled release of aneurotoxin can be increased, but the size of the implant can alsoincrease. As the size of the implant is increased the neurotoxindisperses over a greater area inside of the body, which can decrease theeffectiveness of the implant. In order to avoid this, the implant isencased by a non-permeable material such as titanium. A small opening iskept to allow for pinpoint release of the neurotoxin through the encasedpellet. This effectively can generally allow the implant to havesignificantly different release characteristics. Essentially this canalso allow for thicker section of polymer the neurotoxin will pass,effectively increasing the duration of the neurotoxin release.

[0171] The inner material can be made from a material such aspolyvinylpyrrolidone/methylmethacrylate copolymer. This material allowsfor sustaining a high concentration of neurotoxin complex. Theneurotoxin is complexed with Zn⁺⁺. The complex is then mixed withuncured copolymer, low temperature initiator and an acid (pH between 5.5and 6.8). The mixture is placed into a glass or plastic pellet mold. Themold is placed into a temperature controlled water bath at 35 degrees C.for between about 6 and about 8 hours. This forms the reservoir ofneurotoxin required for a prolonged controlled release.

[0172] In order to prolong the release of the neurotoxin a secondmaterial is then cured around the initial pellet. The pellet (above) isplaced into a mold (insertion molding) with uncured PMMA/low temperatureinitiator. A secondary coating of the uncured PMMA maybe necessary toassure uniform coating of the pellet. Ideally the PMMA thickness is 0.5mm. To form multiple layers, the same insertion molding technique isapplied as described above.

[0173] When the last layer of high density polymer is to be applied, atitanium pellet is used as the mold. The pellet is placed inside of thetitanium pellet with uncured PMMA. The lid to the pellet is secured andthe pellet is placed into a forced air oven at about 35 degrees C. forabout 6 hours to about 8 hours. The lid of the pellet has a 22 gaugeopening to allow for release of the neurotoxin. In this example thepellet effectiveness can be for about 10 months to about 24 months.

Example 5 Neurotoxin Implant With Layered Column

[0174] In order to sustain release for prolonged periods of time analternative approach is to place a layers of the—high densitypolymer/low density polymer w/neurotoxin inside of the titanium pelletdescribed above. Curing can be carried out in a forced air oven at about35 degrees C. for between about 6 hours and about 8 hours for each layerapplied. The diameter of the pellet would be key determinant on theamount of neurotoxin applied. The number of layers can determine howlong the implant will sustain effectiveness. For each layer thethickness of the PMMA layer can be about 0.5 mm and the low densitypolymer w/neurotoxin can be about 0.3 mm. For each layer added, anapproximately 3-month increase in effectiveness is obtained. An implantwith a 2 year life can be made by increasing the length of the implantto about 6.4 mm plus the size of the titanium shell cross section about1 mm for a total of about 7.4 mm.

[0175] Compositions and methods according to the invention disclosedherein has many advantages, including the following:

[0176] 1. a single implant can be used to provide therapeuticallyeffective continuous or pulsatile administration of a neurotoxin over aperiod of one year or longer.

[0177] 2. the neurotoxin is delivered to a localized tissue area withouta significant amount of neurotoxin appearing systemically.

[0178] 3. reduced need for patient follow up care.

[0179] 4. reduced need for periodic injections of neurotoxin to treat acondition, such as a neuromuscular disorder.

[0180] 5. increased patent comfort due to the reduced number ofinjections required.

[0181] 6. improved patient compliance.

[0182] An advantage of our controlled release formulations forneurotoxins include long term, consistent therapeutic levels ofneurotoxin at the target tissue. The advantages also include increasedpatient compliance and acceptance by reducing the required number ofinjections.

[0183] All references, articles, publications and patents and patentapplications cited herein are incorporated by reference in theirentireties.

[0184] Although the present invention has been described in detail withregard to certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe methods of the present invention. Additionally, the presentinvention includes local (i.e. intramuscular, intraglandular,subcutaneous, and intracranial) administration methods wherein two ormore neurotoxins, such as two or more botulinum toxins, are administeredconcurrently or consecutively via implant. For example, botulinum toxintype A can be administered via implant until a loss of clinical responseor neutralizing antibodies develop, followed by administration viaimplant of a botulinum toxin type B or E. Alternately, a combination ofany two or more of the botulinum serotypes A-G can be locallyadministered to control the onset and duration of the desiredtherapeutic result. Furthermore, non-neurotoxin compounds can beadministered prior to, concurrently with or subsequent to administrationof the neurotoxin via implant so as to provide an adjunct effect such asenhanced or a more rapid onset of denervation before the neurotoxin,such as a botulinum toxin, begins to exert its therapeutic effect.

[0185] Our invention also includes within its scope the use of aneurotoxin, such as a botulinum toxin, in the preparation of amedicament, such as a controlled release implant, for the treatment of amovement disorder, and/or a disorder influenced by cholinergicinnervation, by local administration via the implant of the neurotoxin.

[0186] Accordingly, the spirit and scope of the following claims shouldnot be limited to the descriptions of the preferred embodiments setforth above.

We claim:
 1. A controlled release system, comprising: (a) a polymericmatrix, and: (b) a quantity of neurotoxin located within the polymericmatrix, wherein fractional amounts of the neurotoxin can be releasedfrom the polymeric matrix over a prolonged period of time.
 2. Thecontrolled release system of claim 1, wherein neurotoxin is releasedfrom the polymeric matrix in a substantially continuous or monophasicmanner.
 3. The controlled release system of claim 1, wherein theprolonged period of time during which neurotoxin is released from thepolymeric matrix extends over of time of from about 10 days to about 6years.
 4. The controlled release system of claim 1, wherein thepolymeric matrix is comprised of a substance which is substantiallynon-biodegradable.
 5. The controlled release system of claim 1, whereinthe neurotoxin comprises a polypeptide.
 6. The controlled release systemof claim 1, wherein the neurotoxin comprises a presynaptic neurotoxin.7. The controlled release system of claim 1, wherein the neurotoxin is aClostridial neurotoxin.
 8. The controlled release system of claim 1,wherein the neurotoxin is a botulinum toxin.
 9. The controlled releasesystem of claim 1, wherein the neurotoxin is a botulinum toxin selectedfrom the group consisting of botulinum toxin types A, B, C₁, D, E, F andG.
 10. The controlled release system of claim 1, wherein the neurotoxinis a botulinum toxin type A.
 11. The controlled release system of claim1, wherein the polymer which comprises the polymeric matrix is selectedfrom the group consisting of methacrylate, vinyl pyrrolidone, vinylalcohol, acrylic acid, polymethylmethacrylate, siloxane, vinyl acetate,lactic acid, glycolic acid, collagen, and bioceramic polymers andcopolymers thereof.
 12. The controlled release system of claim 1,wherein the quantity of the neurotoxin is between about 1 unit and about50,000 units of a botulinum toxin.
 13. The controlled release system ofclaim 1, wherein the quantity of the neurotoxin is between about 10units and about 2,000 units of a botulinum toxin type A.
 14. Thecontrolled release system of claim 1, wherein the quantity of theneurotoxin is between about 100 units and about 30,000 units of abotulinum toxin type B.
 15. The controlled release system of claim 1wherein the neurotoxin is a botulinum toxin which is released in anamount effective to cause flaccid muscular paralysis of a muscle ormuscle group at or in the vicinity of the implanted system.
 16. Acontrolled release system, comprising: (a) a polymeric matrix, and; (b)between about 10 units and about 20,000 units of a botulinum toxinwithin the polymeric matrix, wherein fractional amounts of the botulinumtoxin can be released from the polymeric matrix over a prolonged periodof time extending from about 2 months to about 5 years.
 17. A method formaking a controlled release system, the method comprising the steps of:(a) dissolving a polymer in a solvent to form a polymer solution; (b)mixing or dispersing a neurotoxin in the polymer solution to form apolymer-neurotoxin mixture, and; (c) allowing the polymer-neurotoxinmixture to set or cure, thereby making a controlled release system. 18.The method of claim 17, further comprising the step after the mixingstep of evaporating solvent.
 19. A method for using a continuous releasesystem, the method comprising injection or implantation of a controlledrelease system which includes a polymeric matrix and a neurotoxin,thereby treating a movement disorder or a disorder influenced bycholinergic innervation.
 20. A method for making a metalcation-complexed neurotoxin comprising the steps of: (a) forming asolution containing a neurotoxin; (b) mixing a multivalent metal cationcomponent with the neurotoxin solution to complex the multivalent metalcation with the neurotoxin, thereby forming a metal cation-complexedneurotoxin suspension, and; (c) drying said suspension to form the metalcation-complexed neurotoxin.